Method of preparing gamma-glutamyl peptides and intermediates



Unit States Patsfi IMETHOD F PREPARING y-GLUTAMYL PEPTIDES ANDINTERMEDIATES Gaston Amiard, Noisy-le-Sec, Rene Heymes, Romain-----.yille,-.;and Leon Velluz, Paris, France, assignors to UCLAF, Paris,France, a corporation of France No Drawing. Filed Dec. 18, 1956, Ser.No. 628,976 Claims priority, application France Dec. 30, 1955 9 Claims.(Cl. 260-112) This invention relates to a new process for preparing 'y-glutamyl peptides by directly reacting an amino acid or peptide esterw1th free N-trityl glutamic acid in the presence of a disubstitutedcarbodiimide.

We found that blocking the glutamic acid by .trityla- 1 tion at thenitrogen permits a selective condensation between the y-carboxyl groupof glutamic acid and the amino group of a-amino acids if this peptidecondensation is carried out in the presence of a di-substitutedcarbodiimide.

It is, therefore, the principal object of the present invention toprovide y-glutamyl peptides of the general.

formula COOH wherein n represents a denominator ranging from 0 to 8 "thepreparation of these y-glutamyl peptides.

These and other objects and advantages of the present invention willbecome more obvious from the herein-following detailed description andfrom the appended claims.

As to the substantial advantages of the herein-claimed "new method ofproducing 'y-glutamyl peptides in comparison to prior methods, attentionis called to our paper in the Bull. Soc. Chim., 1956, p. 97, while thefollowing short discussion is believed to sufiice in connection withthis application. a

.In our copending application Serial No. 596,151 of July 6, 1956, nowPatent No. 2,933,487, a method has been decribed of preparing aand'y-glutamyl peptides from glutamic acid,

HO0C-(l7HCHr-CHz-COOH 7 NH; 7 I According to this copending application,ot-glutamyl "peptides are prepared by reacting an N-tritylated glutamiccu or y-monoester with the ester of an amino acid or peptide inthepresence of dicyclohexylcarbodiimide, followed by alkaline.saponification and detritylation by means of aqueous. acetic acid. l'-Glutamyl peptides are produced 1 by reacting oc-benzylN-tritylglutamate with the benzyl ester of an amino acid or peptide in thepresence of dicyclohexylcarbodiimide and subjecting the resulting N-tritylated derivative of the 'y-glutamyl peptide benzyl diester to aselective hydrogenolysis followed by detritylationl Advantage is therebytaken of the discovery that the: tat-ester group of glutamic acid benzyldiesters is very fstable against alkalies and alcoholysis agents, whichpermits to readily obtain 'y-alkylated a-benzylated diesters andtherefrom the a-benzylated monoester bymonosaponification or the'y-alkylated monoester by hydrogenolysis. When carrying out theafore-described process, itwas noted that only the a-benzyl glutamicester permits the preparation of pure 'y-glutamyl peptides since it doesnot require an alkaline treatment While liberating nt-carboxyl after theformation of the 'y-peptide. Indeed, it was observed that the alkalinetreatment of a diester of N- trityl 'y-glutamyl peptide II, leads to theformation of primarily a-glutamyl peptide IV, most probably over thecyclic derivative III, as illustrated by the following structuralformulae:

wherein R represents an alkyl, aryl or a heterohyclic group, Rrepresents an alkyl and R" represents an'alkyl that may or may not beidentical with R. r

In our copending application Serial No. 628,975, filed December 18,1956, entitled Method of Making Glutathione and Intermediates, nowPatent No, 2,900,375, a new method of producing L- y-glutamylL-eys-teinyl glycine has been describedwherein the last step in thetripeptide synthesis consists in condensing the ethly S-tritylL-cysteinyl glycinate with N-trityl glutamic acid in order to obtain theester of the desired S,N-ditrityl 'y-glutamyl tripeptide. There, theblocking action of the nitrogenbound trityl group of the glutamic acidpermits the selective condensation of the 'y-carboxyl with the aminogroup of the ethyl S-trityl L-cysteinyl glycinate.

Now we have found that the selectivity of the aforedescribedcondensation at the 'y-position occurs also with a-amino acids, whereby,however, the molecular hindrance is much less than in the case of ethylS-trityl L- cysteinyl glycinate. This surprising discovery permits us,

as; set forth infthe herein-claimed invention, to broaden the use ofN-trityl glutamic acid and to obtain 'y-glutamyl peptides without theintermediary formation of the N-tritylated OL-bEl'lZYl glutamic esterobtained as the result of .monosaponification of rat-benzyl 'y-methylN-trityl glutamate prepared according to our copending applicationSerial No. 594,117, filed June 27, 1956, now Patent No.

N-trityl L-glutamic acid is readily obtained by hydrogenolysis ofdibenzyl N-trityl L-glutamate prepared ac- "a disubstituted carbodiimideand an general formula Preparation of amino ester of the new product isobtained in form of small, colorless prisms,

derivatives of such groups, and R represents a'lower alkylsuc'h asmethyl orethyl, permitting the'reaction mixture to remain undisturbed ata temperature ranging fro'rn 0 C. to the refluxing temperature of thesolvent used, eliminating excessive carbodiimide by means of an acidtreatment, saponifying by means'of analkaline treatment, isolating Ntrityl peptide and detritylating the latter by means of heating in anaqueous or alcoholic acid to obtain a compound of the following formula:

COOH

CH-NH2. 7

H2 (CH2) r-Rr 0 O-NH-CH-COOH VIII The method is preferably carried outin the following manner;

N-trityl L-glutamic acid in the form ofthetriethylamine acid saltis.dissolved in methylene chloride, and

the ester of the amino acid which is to be condensed therewith, as wellas an equimolecular amount of dicyclohexylcarbodiimide are added. Thesolution is left standing at room temperature for some time, and thedicyclohexylurea that forms upon treatment with acetic acid isseparated. After the usual washings the solution is 'evaporated todryness and the residue is sap'onifie d by means of an alcoholic sodasolution. N-trityl -glutamyl peptide, VII, is isolated and thendetritylated by heating in aqueous or alcoholic acetic acid.

' EXAMPLE 1 'y-L-glutamyl L-tyrosine (Vlll in connnnsa'rroiv or ETHYLL-TYROSTNATE WITH N-TRITYL L-GLUTAMIC'ACID AND SAPO NIFICATION OF THEESTER GROUP l 8.85 g. of ethyl L-tyrosinate and 3.8 g. ofdicyclohexylcarbodiimide' are dissolved in 50 cc. of methylene chloride,and 8.8 g. of 'triethylamine salt of'N-trityl- L- glutamic acid areadded. The solution is'left standing .overnightat room temperature.After cooling to 0 C.,

dicyclohexylurea, is separated and washed with-10 cc.

of methylene chloride. The filtrate and the methylene chloride washingare combinedian'd washed with 45 cc. of ,N/2 hydrochloric acid andwater, dried overj'mag- .nesium sulfate and vacuum evaporated to drynessat .a temperature'of less than 'C. The residue obtained insoluble inwater, ether, acetone, benzene, chloroform, difiicultly soluble in coldalcohol. It contains two molecules of crystallization ethanol which itloses successively when heated to and C.

Analysis of the product dried at 80 C.: C H O N C H OH= 598.67.Calculated: 70.2% r c; 6.4% H; 18.7% Oj 4.7% N. Found: 70.2% C; 6.3% H;19.1% 0; 4.9% N. I (b) DETRITYLATION OF N-TRITYL "Y'LTGLUTAMYLL-TYROSINE 3.5 g. of N-trityl *y-L-glutamyl L-tyrosine are introducedinto 20 cc. of 50% aqueous acetic acid, and the solution is heated inthe water bath for ten minutes. Triphenylcarbinol is separated andwashed with water and the wash water is combined with the filtrate andvacuum evaporated to dryness at a temperature not exceeding 30" C. Theresidue is dissolved in 6 cc. of water, 200 cc. of acetone are graduallyadded,"and the solution is left standing overnight at room temperature.Filtration and washing with acetone and ether produces 1.7 g. (97%) of"y-L-glutarnyl L-tyrosine', M.P.=265- 270 C.,' [a] P:+26:':2 (c.=2%,water). The product is identical with a sample of ry-L-glutamylL-tyrosine prepared according to G. A niard, R.'Hey-mes and L. Velluz,Bull. Soc. Chim., 1956, p. 97. I

- EXAMPLE 2 Preparati on'of y -L-glutamyl L-leucine p (0) CONDENSATIONOF ETHYL L-LEUCINATE WITH.

N-TRITYL L-GLUTAMIC ACID AND SAPONIFICATION OF THE ESTER GROUP 1.95 g.of ethyl leucinate hydrochloride are dissolved in 3 cc; of'chlo-roform'and the solution is cooled to 0" C. Diethylamine is added drop by dropuntil the pH. is 9,

and 30 cc. of cold sulfuric ether are introduced into the. mixture.

nrethylene chloride, the mixture is first left standing for one hour atroom temperature, and is then treated with "0.5 cc. of acetic acid, andthe dicyclohexylurea that has formed is separated by filtration. Thefiltrate is washed With water, diluted hydrochloric acid, and againwith'water, and is then dried over magnesium sulfate and vacuum evaporated todryness without heating,- For the purpose of saponification,vthe'resulting residue is, dissolved in 5 cc.. of ethanol and 15.4 ccJofnormal soda are added. After heating the mixture for 10 minutesto 70 C.,50 cc. of iced water are added, then, 15.4 cc. of normal hydrochloricacid, and the N-trityl peptide is extracted with chloroform. Thechloroform solution is .washed with water, dried'over magnesium sulfateand vacuum evaporated to dryness. The residue, crystallized in 2.5 cc.of absolute alcohol, yields-pure, hitherto unknown, N-trityl'y-L-glutamyl L-leucine, M.P.=141-144" C., [a] 35-* -2 (c.=2%,chloroform).

Analysis.C H O N =502.59. Calculated: 71.69% C; 6.83% H; 5.57% N. Found:71.4% C; 7.0% H; 5.8% N.

(b) DETRITYLATION OF N-TRITYLq-L-GLUTAMYL L-LEUCINE 2.8 g. of N-trityl-L-glutamyl L-leucine are introduced into 10 cc. of 50% aqueous aceticacid. The solution is heated on a boiling water bath for seven minutesand separated triphenylc'arbinol is removed and washed with water. Thewash water is combinedwith the filtrate and and L. Velluz, Bull. Soc.1956, p. 97. jf'flf r EXAMTLE 3 Preparation of 'y-L-glatamylL-methionine (VIII; n=1, R =CH +CH CCNDENSATION OF THE METHYL ESTER OBLJYIETHIONINE WITH N-TRI'I'YL L-GLUTAMIC ACID AND SAIi'ONIFICATION OFTHE ESTER GROUP 0.9 of the hydrochloride salt of the methyl ester ofL-methionine are dissolved in 3 cc. of chloroform. The solution iscooled to 0 C., diethylamine is added drop by drop until the. pH is 9,and 30 cc. of cold sulfuric ether are introduced. The precipitateddiethylamine 11ydr ochloride is separated and the filtrate is vacuumevaporated to dryness without heating. The residue is taken up with 3cc. ofmethylene chloride. After adding 2.2 g.

.pf the triethylamine saltof N-trityl glutamic acid dissolved in cc. ofmethylene chloride and 0.98 g. of dicyclohexylcarbodiirnide dissolved in4 cc. of methylene chloride, the solution is left standing at roomtemperature for three hours, whereupon it is treated with 0.3 cc. ofacetic acid and filtered. The filtrate is washed with water, dilutedhydrochloric acid and again with water, and is then dried over magnesiumsulfate and vacuum evaplsdrated to dryness without heating. In order tocarry out *saponification', the residue obtained in this manner isdiss'olved in 3 cc. of absolute alcohol, 6.9.cc. of normal .so'daareadded, and the mixture is heated for minutes to 65 C. After adding 30cc. of ice water and 6.9 cc. of normal hydrochloric acid, the mixture isextracted with chloroform. The chloroform solution is washed with water,dried over magnesium sulfate and vacuum evaporated to dryness Thisproduces a residue which, after crystallization in 5 cc. of absolutealcohol, yields pure, hitherto unknown, N-trityl -L-glutamylL-methionine, M.P.=about 140 C. h l

(b) DETRITYLATION OF N-TRITYL 'y-L-GLUTAMYL T 'L-METHIONINE ,520 mg. ofN-trityl 'y-L-glutamyl I .-methionine are siispe'ndedin 2.5, cc. of 50%aqueous acetic acid, and the siispeiisidnisheated for seven minutes in aboiling water .amyl glycinate. 2.45 g. of this salt are dissolved in cc.of water, 6 cc. of normal hydrochloric acid are added,

,bathr After cooling and adding 2 cc. of water, triphenylcarbinol isseparatedby filtration. The filtrate is evaporated, todryness.undervacuum at a temperature notexceeding C. 40 ecfof acetoneare added to the residue,'-;.which is separated, washed with acetone andether and then dried u nder vacuum. Y

This produces 175mg. of "y-L-glutamyl L methionine, M.P.=228-231 C., [u]'-"-'--9-'.:1 (c.=1.4%, water). hitherto described, appears in the formof colorless needles, soluble in water, insoluble in alcohol, ether,acetone, benzene, chloroform.

Analysz's.C H O N S=278.32. Calculated: 43.2% C; 6.5% H; 10.1% N. Found:43.2% C; 6.5% H; 9.8% N.

EXAMPLE 4 Preparation of 'y L-glutamyl glycine (VIII; 11:0, R =H) (a)CONDENSATION OF N-TRITYL L-GLUTAMIC ACID WITH ETHYL GLYCINATE ANDSAPONIFICATION OF THE ESTER This product, which has not been pendedii'i'25 cc ofj inethylenechloride and the suspen sion is cooled to 0 C.Gradually, while stirring, 1 cc. of .die'thylamineis added, then 4.9. g.of the triethylarnine salt of Nrtrityl .L-glutamic acid and.2.1 g. ofdicyclohexylcar'bodiimide. The mixture is left standing overnight at'roomfiemperature and, after adding 0.5 cc. of

acetic acid, dicyclohexylurea isseparated and Washed with 10 cc. .ofmethylene chloride. The filtrate and the wash solutionare combined,first washed with N/2 hydrochloric acid and ice water, and are thendried over sodium sulfate and. vacuum evaporated to dryness. The residueis takenup with 25 cc. of ethyl acetate, 1 cc. of diethylamine is added,andthe solution is left standing for ten .hours inthe' sold. Separationof the solids yields 3.65 g.

(67%) of the diethylamine salt of ethyl N-trityl 'y-L-glutand thesolution is extracted with 50 cc. of ether. The solution is first washedwith ice water, then dried over inagnesiumsulfate and vacuum evaporated.to dryness.

The resulting product (ethyl 'y-N-trityl L-glutamyl glyfcinate) isdissolved in 10 cc. of alcohol. -mal soda are added to the solutionwhich is left standing 10 cc. of norfor one and one-half hours at roomtemperature. After cooling to 0 C., 11 cc. of normal hydrochloric acidare added and the solution is extracted with 25 cc. of chloroform. Thechloroform solution is washed three times with ice water and then vacuumconcentrated to 5 cc.

The "solids are separtaed, washed with chloroform and Detritylation canbe carried out by means of aqueous acetic acid according to the processdescribed at 1(b), 2(b), and 3(b), but may be also accomplished in thefollowing manner: a

l g. of N-trityl 'y-L-glutamyl glycine is dissolved in 9 cc.'of alcohol,:0.9 cc. of acetic acid are added, and the solution is heated for onehour to 65 C. After coolingto room temperature, separating, washing withalcohol a'nd acetone and drying at 90 C., 0.3 g. (66%) of y-L-glutamylglycine are obtained, identical with a sample prepared according to theprocess of G. Amiard, R. Heymesand L. Velluz, Bull. Soc. Chim., 1956, p.97.

Preparation of glutamic N-trityl acid (a) PREPARATION OF DIBENZYLNTRITYL L-GLUTAMATE 18.2. g. of dibenzyl L( glutamate hydrochlorideprepared according to H. Sachs and E. Brand (J. Am. Chem. Soc., 1953,75, 4610) are dissolved in cc. of chloroform. The solution is cooled to0 C.., and 15 cc. of triethylamine are added. While maintaining atemperature of 0 C., 14 g. of trityl chloride are added, and thesolution is left standing at room temperature for forty-eight hours. Thesolution, wherein the triethylamine hydrochloride has partlycrystallized, is washed with water, then with HCl acidified Water andagain with water until the wash water is neutral. The chloroformsolution is dried over magnesium sulfate and evaporated to dryness in awater bath at 40 C. The residue is taken up with 75 cc. of hot absolutealcohol, about 15 cc. of which are distilled off in order to completelyeliminate the chloroform. After the start of crystallization, initiatedby scraping the walls with a stirrer, the flask is cooled and thecrystalline solids separated and Washed with methanol, producing 23.1 g.of dibenzyl N-trityl L-glutamate that can be used directly forhydrogenolysis 1.4 g. of the hydrochloride of ethyl glycinateare sus- 75(yield: 81%).

57g. of dibenzyl N t'rityl'L-g'utamatfe ar e dissolv' -500 cc. of ethylacetate, palladiumon charcoal prepared a HYDROGENOLYSIS OF DrBENzrrN-Irtrmrr 7 .L-GLUTAMAQEE arm from 15 g. of vegetable blackand cc. "of a20% aqueous palladous chloride solution is introducedgland 27 cc. oftriethylamine are added. The'mixture 'is hydrogenated until thetheoreticahamount' ofi-hy'drogen" is absorbed, which requires aboutforty-five minutes. The

catalyst is then separated and washed with ethyl acetate,

the wash water combined with the filtrate is concentrated to about 100cc. After cooling, 100 cc. of .ether are added to-the solution whichisseparated from solids, washed with ether and dried at 80 C.,;yieldin'g40" g. (81%) of the triethylamine acid salt'of: N'-trityl L glutamicacid used in the process: of the "present invention. However, thisN-trityl L-glu'tamic acid'may 'be alsoprepared by directlytritylatin'g'. L-glutarnic acid or by. saponifying an N-tritylL-glutamic ester. .1

It is, of course, possible to change we afore exemplilfifid methods incertain respects. Thus, changes may be made in the solvents, thetemperature or duration of the reaction-without exceeding the sco'p'eofthe hereinclaimed invention. r l

The melting points stated in the examples are instantaneous meltingpoints obtained by means of the heated block method. 1

Weclaim: 1. In a process of producing y-L-glutamyl peptides of theformula e Oil-NH:

T c an,"

wherein X is the radical of an d-amino carboxylic acid linked to theCO-- group or glutamic acid byfits a-amino nitrogen atom, said a-aminocarboxylic acid being an acid occurring in natural peptides andproteins, the steps which comprise preparing aisolu'tionof N-trityl-L-glutamic acid in methylene chloride,wa'dding'to said solutiondicyclohexyl carbodiimide as condensing agent and a lower alkyl ester ofsaid a-amino carboxylic'facid, allowing the mixture'to stand atatemperature ranging from 0 C. to the boiling point of the solvcn tuntil condensation is completed, treating'the reaction mixture groupconsisting of N-trityl- -L-glutamyl-L-tyrosine, N-

carbodiimide as condensing agent and 'alower: allcyl ester of ana-arnino carboxylic acid selected fromqthe group consisting ofL-tyrosine, L-leucine,-L-methionine,

and glycine, allowing the mixture to stand at a temperature ranging from0 C. to the boiling point of the solvent, until condensation iscornpleted, treating the 'reaction mixture of room temperature withdilute acetic acid to destroy unreacted dicyclohexyl carbodiimide,filtering off precipitated dlcyclohexyl urea formed er y, evaporatingthe filtrate to 'dryness, dissolving-the residue in e thanol, adding Nsodium carbonate solution to the resulting solution, heating the mixtureto a temperature ranging from room temperature to C.until'saponification of the ester group is completed, recovering theresulting N-trityl peptide, heating said N-trityl peptide with about 50%aqueous acetic acid until the trityl group is split 'off, filtering offthe precipitated triphenyl carbiriol, and recovering the resulting-L-glutan'iyl peptide from the hydrolysis mixture.

3. The N-trityle'y-L-glutamyl peptide of the formula 06H. coonoarrr-o-Nn in 0.11., 1 torn wherein X is the radical of an a-aminocarboxylic acid I linked to the -CO group of glutamic acid by .its

a'-amino nitrogen atom, said a-amino carboxylioa'cid being an acidoccurring in natural peptides and proteins. 4. The N-trityl-y-L-glutamylpeptide selected from the .9; 'y-L-glutamyl L-methionine p I ReferencesCited in the tile this patent Benary et al.: Berichte, vol. 57 (1924).pp; 1324 21.

. Helferich et .al.: Berichte Deut. ChemgCeseL,

58 Pp- 87 3-875, 8826 (1925).. I v V v .Anson ,et al.: Advancesin-Protein Che i vol? 5 pp- 2 -32, 43-44 and 7 2.. r r

Arniard et al. un. Soc. Chim. (FranceflNlo'lf 2111955) Sheehan et at:J.A.C.S., v61. '70 1955 lll6lf8 Noller: Chemistry of Org. Compounds, 2nded., 19 58'-, pp..305-6. Y l

1. IN A PROCESS OF PRODUCING $-L-GLUTAMYL PEPTIDES OF THE FORMULA